Polylactic acid, which is a biodegradable polymer, draws strong attention as a product responding to sustainability and LCA (life cycle assessment) with actualization of CO2 problem and energy problem, and an efficient and economical production method is required for lactic acid, which is a raw material thereof.
Incidentally, polylactic acid produced industrially currently is an L-lactic acid polymer. However, lactic acid includes L-lactic acid and D-lactic acid. D-lactic acid is calling attention in recent years as a raw material for polymers or intermediates of agricultural chemicals and medicines. However, lactic acid as a raw material is required to have high optical purity for any use.
In the nature, there exists a microorganism which produces lactic acid in good efficiency such as lactobacillus and filamentous bacteria, and some of methods for producing lactic acid using them have already come into practical use. For example, Lactbacillus delbrueckii or the like is known as a microorganism producing L-lactic acid in good efficiency, and microorganisms belonging to the genus Sporolactobacillus or the like as microorganisms producing D-lactic acid in good efficiency. In all cases, the amount of lactic acid accumulated reaches high level, but by-products contained in the culture solution other than lactic acid, for example, compounds such as acetic acid, ethanol, acetoin and pyruvic acid, which are not removed in the purification process, may lead to decrease in the quality of lactic acid, which is the final product. In addition, it is also a critical problem that contamination due to optical isomers can cause decrease in optical purity.
To avoid such decrease in the purity of lactic acid, it is an effective means to decrease the amount of by-products produced by a microorganism. It has become possible to inhibit specifically production of the aimed by-products by disruption of a certain gene of a microorganism using a gene recombination technique which has been developed in recent years. However, it does not mean that the gene disruption method can be easily applied to any microorganism in reality, and it is not easy to apply it to a microorganism which can originally produce lactic acid in high yield such as lactobacillus or filamentous bacteria or the like. It is because genome information of these microorganisms cannot always be known sufficiently, and further they are not universally used as a host of gene recombination.
In contrast, it is possible to perform gene disruption relatively easily for Escherichia coli, yeast, human culture cell or the like, for which genome information is abundant, and which have sufficient records as a host of gene recombination. Especially, Escherichia coli is most preferred in view of growth rate or easiness of culturing. Furthermore, since Escherichia coli produces only D-isomer of lactic acid, it is a suitable host for the purpose of obtaining D-lactic acid with high optical purity. However, wild type of Escherichia coli has low productivity for D-lactic acid, and produces a variety of by-product organic acids in addition to D-lactic acid. To solve this problem, it has been tried in the past to modify metabolic pathway of Escherichia coli by gene recombination, for selectively producing D-lactic acid in high yield.
Chang, et al. (Chang, D.-E., et. al., Appl. Environ. Microbiol., Vol. 65(4), pp 1384-1389 (1999)) were able to produce 62.2 g/L of D-lactic acid in 60 hours, by culturing a double variant of phosphotransacetylase (hereinafter, may be simply referred to as pta) and phosphoenolpyruvic carboxylase (hereinafter, may be simply referred to as ppc) of Escherichia coli using a medium containing 5% glucose and amino acids and after increasing the amount of microbial mass by preliminary culturing under aeration, culturing was performed under anaerobic conditions, and by further culturing and adding glucose to the medium so as to maintain glucose concentration at 5% or less in the medium. In this case, the conversion rate from glucose to D-lactic acid was 76%.
Zhou, et al. (Zhou, S., et. al., Appl. Environ. Microbiol., Vol. 69(1), pp 399-407 (2003)) reported that Escherichia coli quadruply disrupted in pyruvate formate-lyase (hereinafter, may be simply referred to as pfl), fumarate reductase (hereinafter, may be simply referred to as frd), alcohol/aldehyde dehydrogenase (hereinafter, may be simply referred to as adeE) and acetate kinase (hereinafter, may be simply referred to as ackA) was prepared and cultured in an inorganic salt medium containing 5% glucose under anaerobic conditions for 168 hours to produce 48.5 g/L of D-lactic acid with no by-product of formic acid, succinic acid, ethanol and acetic acid. However, this attempt cannot be said to satisfy both of selection rate and productivity because of low productivity such as 0.29 g/L/hr although it was successful for producing D-lactic acid with high selectivity. In addition, there is no mention about by-product pyruvic acid, and effects of its decrease are unclear. Since pyruvic acid is a metabolic reaction substrate of D-lactic acid, it is different from other by-product organic acids, and if its production is suppressed thoughtlessly, production of D-lactic acid itself is suppressed as well. In that point, it is not easy to suppress by-production of pyruvic acid to a minimum. Generally, it is a well-known fact to a person skilled in the art that when pyruvic acid is contained as an impurity in a raw material for lactic acid monomer, unwanted problems occur such as decrease in polymer polymerization rate or the like. For this reason as well, pyruvic acid is one of by-products that should be decreased by all means. However, there has been no report in the past that by-production of pyruvic acid is suppressed while maintaining high productivity of D-lactic acid successfully.
In summary, the maximum amount of D-lactic acid accumulated with Escherichia coli known until now has been 62.2 g/L with the production time of 60 hours. On the other hand, if it is considered that L-lactic acid productivity of lactobacillus or filamentous bacteria, which is used in industrial production of L-lactic acid, is not less than 100 g/L in the amount accumulated and further the production time is within 24 hours, it would have to be said that the amount of D-lactic acid accumulated and the production time in the case of Escherichia coli are still at low level. There was no report in the past that Escherichia coli achieved lactic acid productivity as much as that of lactobacillus or filamentous bacteria, and far from it, there was no data in the past which suggest whether or not D-lactic acid can be accumulated and produced exceeding 100 g/L using Escherichia coli. 
When D-lactic acid is fermented using Escherichia coli, the presence of oxygen is generally considered not to be preferable. It is because, if an electron acceptor such as oxygen is present, Escherichia coli undergoes breathing and not fermentation. Only in the absence of an electron acceptor such as oxygen, Escherichia coli gains energy (ATP) only by phosphorylation at substrate level, and produces reductive organic acids such as lactic acid using reduction power (NADH) obtained in the glycolytic pathway. For reasons such as these, conventional D-lactic acid fermentation using Escherichia coli is carried out mostly in anaerobic culture. In the rare case, bi-phasic culture is carried out wherein the first half of the culture is aerated and the second half of the culture is anaerobic. However, this means that the first half is performed for the purpose of ensuring a sufficient amount of microbial mass, and the final lactic acid fermentation is carried out under anaerobic conditions as expected. However, when actual industrial production is supposed, corn steep liquor (hereinafter, may be simply referred to as CSL) or the like, which is a cheap amino acid source added to a medium, contain not only organic acids which become impurities, but also both of D-isomer and L-isomer of lactic acid. However, if anaerobic culture is carried out, L-lactic acid is not assimilated and remains in the medium. L-lactic acid dehydrogenase (hereinafter, may be simply referred to as lld), which is a catalytic enzyme of the reaction to produce pyruvic acid from L-isomer, is known to be expressed under aerobic conditions. Therefore, if there is a method to ferment lactic acid in good efficiency even under aerobic conditions, it is expected that the method would enable to assimilate L-isomer contained in the medium into microbial mass, and to produce D-isomer with high optical purity. However, there has been no technique so far to realize it.
Before of the report by Zhou, et al. about lactic acid production using a pfl disrupted strain, there have been the following reports. Specifically, Contag, et al. (Contag, P. R., et. al., Appl. Environ. Microbiol., Vol. 56(12), pp 3760-3765 (1990)) have shown that, while pfl non-variant Escherichia coli strain produces 35 mM of lactic acid, a pfl variant strain produces 45 mM of lactic acid due to improved productivity of lactic acid. Namely, it has been already known by disclosure of data by Contag, et al. that D-lactic acid productivity is improved by inactivation of pfl activity in Escherichia coli. 
D-lactate dehydrogenase is classified into NADH-dependent one and FAD-dependent one according to the difference in the dependence on a coenzyme. NADH-dependent D-lactate dehydrogenase catalyzes a reaction from pyruvic acid to D-lactic acid in the living body. Especially, Escherichia coli-derived NADH-dependent D-lactate dehydrogenase is called ldhA.
Yang, et al. (Yang, Y. T., et. al., Metab. Eng., Vol. 1(2), pp 141-152 (1999)) have reported that, by introducing an expression vector incorporated with ldhA gene into Escherichia coli, the amount of D-lactic acid accumulated is improved, albeit small, to about 8 g/L. That is, it is known from data disclosed by Yang, et al. that D-lactic acid productivity is improved by reinforcement of ldhA activity in Escherichia coli. 
On the other hand, according to Bunch, et al. [Bunch, P K., Microbiology, Vol. 143(Pt 1), 187-195 (1997)], it has been reported that Escherichia coli, into which an expression vector of Escherichia coli-derived ldhA gene is introduced, is inhibited in growth thereof by the introduced expression vector.
In addition, examples of over-expression in Escherichia coli of D-lactate dehydrogenase (hereinafter, may be called ldh) derived from bacteria other than Escherichia coli, include expression of Lactobacillus helveticus-derived ldh reported by Kochhar, et al. [Kochhar, S., Eur. J. Biochem., (1992) 208, 799-805], and expression of Lactobacillus bulgaricus-derived ldh reported by Kochhar, et al. [Kochhar, S., Biochem. Biophys. Res. Commun., (1992) 185, 705-712]. However, in any of the reports, there is no mention about the amount of D-isomer or of pyruvic acid accumulated, since these reports were concerned with investigation on physicochemical properties of the expressed enzymes.
However, D-lactic acid productivity of a microorganism which is inactivated or decreased in pfl activity, and further reinforced in ldhA activity have not been still well known.
With a method for reinforcing a gene using an expression vector, troubles may happen generally such as loss of the vector causing decrease in the amount of a desired gene expressed, and further decrease in the productivity of a desired substance. From these findings, the method for reinforcing an ldh gene using an expression vector has several problems to be solved in application to industrial production of D-lactic acid, and a method for reinforcing a gene is desired to replace it. However, no report has been made about such approach.
As a method for reinforcing a gene instead of using an expression vector, there is a method for reinforcing the gene wherein the gene promoter region on the genome is substituted by an arbitrary promoter as reported by Solem, et al. (Solem, C., et. al., Appl. Environ. Microbiol., Vol. 68(5), pp 2397-2403 (2002)). However, if a case is considered in which this technique is applied to production of D-lactic acid in which the above-mentioned ldhA gene is used, the ldhA gene reinforced with this method is only 1 copy of the gene on the genome. Thus, it is predicted that ldha activity improvement is small as compared with a reinforcement method by an expression vector which expresses many copies of the gene, so it has been difficult even for a person skilled in the art to anticipate that the D-lactic acid productivity improves as compared with the case of using the expression vector.
On the other hand, it has been disclosed by an analysis of enzyme purified from Escherichia coli that FAD-dependent D-lactate dehydrogenase (hereinafter, may be simply referred to as dld) catalyzes mainly the reverse reaction of NADH-dependent D-lactate dehydrogenase, i.e., the reaction from D-lactic acid to pyruvic acid. Shaw, et al. acquired in the past Escherichia coli strains JS150 and JS151, wherein dld was disrupted. However, there was no mention about D-lactic acid productivity or pyruvic acid productivity in those strains (Shaw, L., et. al., J. Bacteriol., Vol. 121(3), pp 1047-1055 (1975)). In addition, Barnes, et al. have reported that dld is involved in incorporation of a variety of amino acids or saccharides (Barnes, E. M., et. al., J. Biol. Chem., Vol. 246(17), pp 5518-5522 (1971)]. However, there was no mention about D-lactic acid or pyruvic acid production.
In addition, if a search is conducted for the double variant of did and pfl with the database of Yale university-affiliated E. coli Genetic Stock Center (CGSC), which is one of authorities for delivery of an Escherichia coli strain, it gives an article by Mat-Jan, et al., (Mat-Jan, F., et. al., J. Bacteriol., Vol. 171(1), pp 342-348 (1989)] as the corresponding item. However, as the results of actual close examination, there was no description about disrupted double strain of dld and pfl in this article.
As described above, there has been no report for D-lactic acid to indicate achievement of productivity and selectivity at the same time corresponding to industrial level by fermentation production using a microorganism, and also there were no previous examples in which, for example, succinic acid or fumaric acid, which is a main by-product organic acid, was reduced while maintaining high D-lactic acid productivity.
Thus, we have intensively studied in order to suppress succinic acid production without decreasing D-lactic acid productivity, and as a result have found that, by disruption of gene of malate dehydrogenase (hereinafter, may be called mdh), which is an enzyme catalyzing a reaction from oxalacetic acid to malic acid under anaerobic conditions, it is possible to suppress completely production of succinic acid without decreasing D-lactic acid productivity. However, since fumaric acid was still produced as a by-product, we have also found that it is possible to decrease the amount of by-product of fumaric acid by disruption of the gene of aspartate ammonia-lyase (hereinafter, may be called aspA).
An inactivation effect of mdh activity has been disclosed in the article of Courtright, et al. in 1970 (Courtright, J. B. et. al., J. Bacteriol., Vol. 102(3), pp 722-728 (1970)). The disclosed findings indicate that, with Escherichia coli wherein mdh activity is inactivated, while there is no reactivity from oxalacetic acid to malic acid under anaerobic conditions, reactivity from aspartic acid to fumaric acid is improved in contrast. That is, this explains that there are two pathways to produce succinic acid under anaerobic conditions, i.e., a pathway to lead to fumaric acid and succinic acid via malic acid from oxalacetic acid, and a pathway to lead to fumaric acid and succinic acid via aspartic acid from oxalacetic acid, and that if mdh activity is inactivated, the former pathway comes to a stop, but the latter pathway is activated in contrast. Therefore, the article of Courtright, et al. is not a disclosure showing that succinic acid is not produced by inactivation of the mdh activity.
Another prior art on the effects of inactivation of mdh activity relates to yeast in which the mdh gene is disrupted (JP-A No. 11-056361). This patent relates to change of the amount of malic acid produced by disruption of the mdh gene of the yeast, but has no mention on how such results affect the amount of succinic acid produced.
In summary, it has been difficult even for a person skilled in the art to assume from the findings in the past that succinic acid production can be completely suppressed by inactivation of mdh of a microorganism.
In addition, concerning the effect of inactivation of aspA activity, only findings on Yersinia pestis have been disclosed in the past (Dreyfus, L. A., et. al., J. Bacteriol., Vol. 136(2), pp 757-764 (1978)). However, the gist of the present article is that aspartic acid or glutamine becomes less susceptible to decomposition in the cell by inactivation of aspA activity, and there is no discussion about the amount of fumaric acid produced.    Patent Document 1: JP-A No. 11-056361    Non-Patent Document 1: Chang, D.-E., et. al., Appl. Environ. Microbiol., Vol. 65(4), pp 1384-1389 (1999)    Non-Patent Document 2: Zhou, S., et. al., Appl. Environ. Microbiol., Vol. 69(1), pp 399-407 (2003)    Non-Patent Document 3: Contag, P. R., et. al., Appl. Environ. Microbiol., Vol. 56(12), pp 3760-3765 (1990)    Non-Patent Document 4: Yang, Y. T., et. al., Metab. Eng., Vol. 1(2), pp 141-152 (1999)    Non-Patent Document 5: Bunch, P. K., et. al., Microbiology, Vol. 143(Pt 1), pp 187-195 (1997)    Non-Patent Document 6: Kochhar, S., et. al., Eur. J. Biochem., Vol. 208(3), pp 799-805 (1992)    Non-Patent Document 7: Kochhar, S., et. al., Biochem. Biophys. Res. Commun., Vol. 185(2), pp 705-712 (1992)    Non-Patent Document 8: Solem, C., et. al., Appl. Environ. Microbiol., Vol. 68(5), pp 2397-2403 (2002)    Non-Patent Document 9: Shaw, L., et. al., J. Bacteriol., Vol. 121(3), pp 1047-1055 (1975)    Non-Patent Document 10: Barnes, E. M., et. al., J. Biol. Chem., Vol. 246(17), pp 5518-5522 (1971)    Non-Patent Document 11: Mat-Jan, F., et. al., J. Bacteriol., Vol. 171(1), pp 342-348 (1989)    Non-Patent Document 12: Courtright, J. B. et. al., J. Bacteriol., Vol. 102(3), pp 722-728 (1970)    Non-Patent Document 13: Dreyfus, L. A., et. al., J. Bacterial., Vol. 136(2), pp 757-764 (1978)